The present invention relates generally to an apparatus for, and a method of, increasing the quantity of information obtained from flow analyzers or other data collection devices by temporarily storing data relating to, for example, particles, cells, and inter-event background noise from flow through an examination zone. In particular, the present invention relates to an apparatus for and a method of achieving zero dead time by temporarily storing such data in a circular buffer, or similar buffer or temporary storage capability, for later processing.
Flow cytometry is conveniently used to quickly measure one or more optical parameters of the cells and/or particles that pass through a light beam that impinges on a narrow examination zone. Background information on flow cytometry is found, for example, in Shapiro""s Practical Flow Cytometry, Third Edition (Alan R. Liss, Inc. 1995), incorporated herein by reference in its entirety.
In conventional flow cytometers, sample fluid containing sample cells and/or particles is introduced from a sample tube into or near the center of a faster flowing stream of sheath fluid, which draws the sample toward the center of the combined streams. This process, known a hydrodynamic focusing, allows the cells and/or particles to be delivered reproducibly to the center of the measuring point in the flow cell or other examination zone. Typically, the cells and/or particles are in suspension in the flow cell.
A continuous wave laser focuses a laser beam on the cells and/or particles as they pass through the examination zone by a flow of a stream of the suspension. When an object of interest in the flow stream is struck by the laser beam, certain signals result and are sensed by detectors. For example, these signals include forward light scatter intensity, which provides information concerning the size of individual cells and/or particles, and side light scatter intensity, which provides information regarding the relative size and refractive properties of individual cells and/or particles.
Other signals which may be sensed by detectors include fluorescence emissions from one or more fluorescent dyes and/or other fluorescent molecules, for example, tryptophan or other fluorescent amino acid(s) or other molecule(s) that is native to a protein or other peptide, or to another biomolecule or man-made molecule. Typically, when a plurality of different fluorescing molecules is employed in an analytical scheme, fluorescence emission peaks are selected to minimize or, ideally, eliminate spectral overlap between the respective fluorescence emission peaks.
For example, ideally, fluorescence emission peaks will differ by 50 xcexcm, though lesser (or larger) spectral separation also can be accommodated and used to advantage, for example, 20, 30 or 40 xcexcm, where the greater the spectral separation, the more powerful is the discrimination between the respective fluorescence emitters. In addition, quantum efficiency is considered in the choice of fluorescent molecules. In the case of use of a plurality of different fluorescent molecules, a separate detector is tuned to and used for the different wavelength of emission of each fluorescence emitter. The excitation wavelength for more than one fluorescent molecule can be the same, or different excitation wave lengths can be used to match the excitation spectrum of each different fluorescent molecule.
The optical signals that are generated by an analytical procedure are transmitted to an output meter(s) and/or data storage means. Signal processing, for example, by a Digital Signal Processor or DSP, can be employed before and/or after intensity data on the measured optical signals are transmitted to the output meter(s) and/or data storage means.
In flow cytometry, an xe2x80x9ceventxe2x80x9d occurs when a cell or particle passes through the beam of the laser or other light source. As the event progresses, the light measured from scattering and/or fluorescence emission increases as the cell or particle enters the beam, reaches a maximum at the center of the beam, and tapers to a nominal value as it leaves the beam.
State of the art flow cytometers commonly use one of two systems to measure events. One system uses peak detectors, including peak hold circuits, to sustain the maximum signal level obtained from an event, and to measure optical parameters relating to cells and/or particles passing through a laser beam. Once a peak detector has sensed and measured an event, the peak detectors are turned off to provide sufficient time for an analog to digital, or A/D, converter to digitize that maximum value of the signal. During the time that the peak detectors are off, termed xe2x80x9cdead time,xe2x80x9d any event occurring within the laser beam will go undetected.
The second system uses an integrator to measure the area under the peak collected for an event. With either system, when an event occurs within the cytometer""s light beam during the dead time, the event will go undetected.
Normally, such lack of detection of an event does not constitute a significant problem because, for a large number of events, only a tiny number will be missed. However, applications which require the detection of rare, critical events, for example, identification of one special cell or particle in a thousand or a million, suffer from the possible non-detection of a rare event when current generation flow cytometers are used for detection and measurement. The higher the throughput, the greater is the probability that such a critical event will be missed per unit time period.
One solution to problems encountered in sorting samples in flow cytometry is provided by U.S. Pat. No. 5,550,058 to Corio, et al., incorporated herein by reference. Corio, et al. provides a means of flexibly controlling decisions on the sorting of events detected in a flow cytometer. Events are pre-qualified according to user selectable parameters to permit reduction of events missed during dead time. However, the Corio, et al. reference does not teach or suggest a means of reducing the probability of missing a dead time event to zero.
Thus, it would be beneficial in the art of flow analyzers, including flow cytometers or other analyzing equipment, to have a means to reduce to zero the probability of missing any event, including a rare, dead time event.
It is also desirable to efficiently and inexpensively provide a system and/or method of reducing the probability of missing an event.
It is further desirable to efficiently and inexpensively provide a system and/or method of reducing the probability of missing an event for high sampling rates, for example, on the order of approximately a sampling period, such as one sample per millisecond or shorter period.
Accordingly, it is a feature and advantage of the present invention to provide an apparatus and a method for reducing the dead time to zero in flow analyzers, flow cytometers, and other measurement devices.
It is another feature and advantage of the present invention to provide a means to reduce the dead time to zero in flow analyzers, flow cytometers, and other measurement devices by use of, for example, a circular buffer to store optical data until the data can be processed by at least one digital signal processor or other processor.
It is a further feature and advantage of the present invention to provide a means to reduce the dead time to zero in flow analyzers, flow cytometers, and other measurement devices by providing software that permits the user to specify one or more of data collection, data storage, and data processing parameters, such as size of data storage areas, number of data storage areas, sampling rate (and/or the inverse: length of sampling or sample period), signal-to-noise (S/N) threshold, and fixed trailing distance.
It is a feature and advantage of the present invention to efficiently and inexpensively provide a system and/or method of reducing the probability of missing an event.
It is another feature and advantage of the present invention to efficiently and inexpensively provide a system and/or method of reducing the probability of missing an event in the presence of a high sampling rate.
It is yet another feature and advantage of the instant invention to provide the capability of enhancing data analytical capability by going back in time to re-examine and reprocess data in xe2x80x9coldxe2x80x9d data storage areas in order, potentially, to recreate a more complete event peak(s) by applying special algorithms to subthreshold data.
The instant invention provides an apparatus and a method of use of that apparatus to reduce dead time to zero or substantially zero in a measurement device. The apparatus includes 1) a circular buffer that has a) a plurality of data storage areas that are linked to receive and consecutively store incoming data from a plurality of successive sampling periods of a measurement device that measures events occurring in a measurement zone, b) a First Pointer (or first logical pointer), and c) a Second Pointer (or second logical pointer); and 2) at least one Digital Signal Processor (DSP) or other data processor.
Each data storage area is configured to receive and store data from one sampling period. Furthermore, the data storage areas are linked in an order that provides storage for data from a next-in-time sampling period into the next-in-order data storage area. The First Pointer directs receipt and storage of data of the next-in-time sampling period into the next-in-order data storage area; and, once all data storage areas contain data, the First Pointer directs receipt of data of the next-in-time sampling period, and storage by overwriting therewith the data in the next-in-order data storage area, which contains the oldest data of the circular buffer.
The Second Pointer is directed to the data storage area that stores data from a sample period preceding the current time by at least a fixed trailing distance, referenced to the First Pointer; and the Second Pointer directs one of the at least one DSPs to read and process data in the data storage area to which the Second Pointer is directed.
Once the next-in-time data from the current sampling period have been stored, the First Pointer advances to the next-in-order data storage area. In addition, once data in the data storage area to which the Second Pointer is directed have been processed, the Second Pointer advances to the next-in-order data storage area to repeat the cycle of data processing, followed by advancement to the next-in-order data storage area.
Other addressable buffers can alternatively be used where the addressable range of data allows the system to go forward and sufficiently backward to obtain previously collected samples. For example, a FIFO and backward cache combination can alternatively be used in the present invention, as can a cascading buffer.
The method of the instant invention entails applying the above apparatus to collect and process data from events occurring in a measurement device, particularly a measurement device with a flowcell, such as a flow cytometer or other flow analyzer.
The fixed trailing distance is set by the user to equal the longest possible time for an event, or longer, often falling in the 10 to 100 millisecond range. The size and number of data storage areas in the circular buffer also are user specified. Data storage areas often are set to 12 to 16 bits, though larger and smaller sizes may be used to adjust for particular applications. The number of data storage areas often is set to about one thousand, or greater. However, numbers of data storage areas less than a thousand also can be used advantageously.
The sampling rate, and/or its inverse which is length of sampling period, also are user specified. A typical sampling rate is one million per second, which equates to sampling periods of one millisecond each. However, larger and smaller figures can be utilized according to the particular application.
When a plurality of DSPs is used, the user may specify the order of use of the respective DSPs. For example, one DSP can control storage of data incoming to the circular buffer, and one or a plurality of DSPs can handle processing of the data, as directed by the Second Pointer.
Buffers that are functionally equivalent to circular buffers also can be used, for example, a First In-First Out buffer which temporarily stores data (or lack thereof) from the respective data storage areas into one or more caches for possible later processing; double or triple cascading buffers; etc.
In another embodiment, an FPGA (Field Programmable Gate Array) may be programmed as a state machine, for example, with four or five states, to replace use of a DSP for inputting data into the circular buffer or functional equivalent.
The above broad outline of the more important features of the instant invention has been presented 1) to facilitate understanding of the following sections, which provide greater detail with respect to components of the instant invention, and 2) to succinctly highlight contributions of the instant invention to the art. However, it is to be understood that the above outline, as well as the terminology and details as to construction, arrangement and practice of the instant invention presented in the following sections, are exemplary and not limiting; i.e., the instant invention is capable of other embodiments as to construction, arrangement and practice.
As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, can readily be utilized as a basis for designing other structures and methods of practice to execute the purposes of the instant invention. Therefore, it is important that the instant disclosure and claims be regarded as embracing equivalent constructions and practices insofar as they do not depart from the spirit and scope of the invention as disclosed herein.
Another purpose for this SUMMARY OF THE INVENTION section is to enable the U.S. Patent Office and the public generally, including scientists, engineers and practitioner in the art who are not familiar with patent or legal terms or phraseology herein, to quickly determine the nature and essence of the instant disclosure with only cursory review. Thus, the intention of this section is not to limit the scope of the instant invention in any way.